The document analyzes antigenic variation in lngA, the pilin subunit of the CS21 pilus found in enterotoxigenic E. coli (ETEC). It screened 158 ETEC strains isolated from the Middle East, finding 126 to be ETEC-positive. Of these, 41 strains were CS21-positive. Sequence analysis of lngA alleles revealed four groups with variation clustered in the αβ-loop and D-region. Further screening of additional strains is needed to characterize the antigenic variation and evaluate lngA's potential as a vaccine target.

1. Antigenic Variation of LngA, the Structural Subunit of CS21
Nishith Nagabhushana, Pennsylvania State University
Mentored by: CAPT. Stephen Savarino & Ms. Annette McVeigh, Naval Medical Research Center
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Introduction:
Enterotoxigenic Escherichia coli (ETEC) is a predominant cause
of diarrhea in travelers and in young children in developing
countries. ETEC strains are characterized by their ability to
produce heat-labile (LT) or heat-stable (ST) enterotoxins.
Plasmids encoding the enterotoxins are also responsible for
encoding fimbrial colonization factors (CF) which are responsible
for intestinal binding thus making them a possible vaccine target.
A type IV pilus, CS21, is commonly harbored by ETEC and has
been suggested as an efficient vaccine target. However, there is
limited information available to date on the antigenic variation of
lngA, the pilin subunit of CS21. Our focus is to further define the
extent of allelic and predicted antigenic variation in CS21 from a
collection of ETEC strains isolated in the Middle East.
Materials and Methods:
1. ETEC Strain Collection
- Operation Desert Shield/Storm ETEC strains (n=51) derived from
U.S. service members deployed to Saudi Arabia and Egypt (1989-
1990) with ETEC diarrhea.
- Egyptian pediatric ETEC strains (n=97) derived from young Egyptian
children with diarrhea in the second year (1996-7) of a longitudinal
study of community-based diarrhea.
2. Screening for toxins:
- The 158 E. coli strains were screened by PCR to confirm the
presence of toxins. The positive control was H10407 E. coli strain that
has been confirmed an ETEC for all 3 toxin types (LT, STh, STp).
- The following are the PCR cycling conditions and primers used2:
94°C - 5min
94°C – 30sec
58°C – 30sec 25 cycles
72°C - 45sec
72°C – 7min
4°C - hold
3. Screening for lngA gene among the confirmed ETEC strains:
PCR was used to screen for lngA genes with E9034A E. coli strain as
the positive control and the following cycling conditions and primer2:
95°C - 5min
95°C – 30sec
55°C – 30sec 30 cycles
72°C - 1min
72°C – 7min
4°C - hold
4. Sequencing:
The purified lngA PCR product was sequenced using the BigDye
Terminator v3.1 cycle sequencing kit on ABI 3130xI Genetic Analyzer.
5. Sequence Analysis and Phylogram generation:
- The sequences were analyzed using Sequencher 5.1 and assembled
contigs generated a new consensus sequence.
- MacVector version 12.5.1 was used to generate the amino acid
sequences.
- The phylogram tree was generated using an online tool available at
http://www.phylogeny.fr/ using the radial (Drawtree) tree style option.
- Clustal W Allignment tool was used to align the sequences and
develop a schematic view depicting regions of allelic variations in the
mature linear lngA protein.
Primer
Name
Target
gene
Sequence
estA2-4F estA2-4 AATTGCTACTATTCATGCTTTC
GAC
estA2-4R estA2-4 TCTTTTTCACCTTTCGCTCAGG
STpF1 estA1 ATGAAAAAGCTAATGTTGGCA
STpR1 estA1 TTAATAACATCCAGCACAGGCA
LThF1 eltA1
CATAATGAGTACTTCGATAGAG
C
LThR1 eltA1
Primer Name Sequence
out lngA F GGAAGGGAACCGGAAAGATTG
out lngA R CGACGTGCCTCATCAGCTTC
148 E. coli strains
(51 Desert Shield +
97 Abu-Homos )
126 were
confirmed as
ETEC
positive
57 (ST)
15 (lngA
positive)
22 (LT)
0 (lngA
positive)
47 (LTST)
26 (lngA
positive)
Fig 1. A
flowchart
indicating
the
proportion
of LT, ST
and LTST
strains and
their
association
with lngA.
Results:
- To date, 85% of the archived ETEC strains were confirmed to be ETEC positive among which
17% were LT only, 46% ST only and 37% were both LT and ST positive.
- It was observed that 33% (41/126) of the confirmed ETEC strains were CS21 positive of which
26% were associated with ST only, 74% with LTST and none with LT only toxin.
- lngA was dominantly found to be associated with CFA II (CS1, CS2 and CS3).
- Phylogenetic analysis of the predicted amino acids revealed that the lngA alleles fall into 4
different branches as previously published by Gomez et al.1
- Statistical analysis of the data shows that group 1 (reference strains B7A and 11381A) is
dominated by CS2CS3 colonization factor, whereas group 2 (reference strains B2C and M526C6)
is dominated by CS1CS31.
- The CLUSTAL W Alignment of amino acid sequences showed that the variation was mostly
confined to the αβ-loop and D-region that form the globular domain and contain residues that are
important in pilin function.3
Discussion:
Sequence analysis of an expanded number of CS21 lngA genes showed that there is no more allelic and
antigenic variation than previously observed by Gomez et al.1 We plan to screen an additional 200 strains to
verify the nominal antigenic variation which would favor the suitability of lngA as a vaccine target.
Fig 2. A
pictorial
representation
of the
proportion of
lngA positive
strains
associated with
various
colonization
factors.
CS1CS3
25%
CS2CS3
34%
CS6
12%
CFA1
12%
CS3
7%
Others
10%
Fig 3. A phylogenetic tree showing the two groups based on the
reference strains and the proportion of lngA strains associated with
various colonization factors1.
Group 1c
(11381A)
n=21 alleles
Group 1a
(10159A)
n=1 alleleGroup 1b
(B7A)
n=7 alleles
Group 2a
(B2C)
n=14 alleles
Group 2c
(M526C6)
n=3 alleles
Group 2b
(E9034A)
n=1 allele
Acknowledgements:
Dr. Tony Vortherms, Walter Reed Army Institute Of Research
Dr. Steven Poole, Naval Medical Research Center
Ms. April Fordyce, Naval Medical Research Center
References:
1. Oscar G. Gomez-Duarte, Sujay Chattopadhyay, Scott J. Weissman, Jorge A. Giron, James B. Kaper, Evengi V.
Sokurenko, “Genetic Diversity of the Gene Cluster Encoding Longus, a Type IV Pilus of Enterotoxigenic
Escherichia coli.” Journal Of Bacteriology , Dec 2007, p. 9145-9149.
2. Rania A. Nada, Hind I. Shaheen, Iman Touni, Dina Fahmy, Adam W. Armstrong, Matthew Weiner, John D Klena,
“Design and validation of a multiplex polymerase chain reaction for the identification of enterotoxigenic Escherichia
coli and associated colonization factor antigens.” ScienceDirect, 2010, p. 134-142.
3. Subramaniapillai Kolappan, Justin Roos, Alex S. W. Yuen, Owen M. Pierce and Lisa Craig, “Structural
Characterization of CFA/III and Longus Type IV Pili from Enterotoxigenic Escherichia coli.” Journal of Bacteriology,
May 2012, p. 2725-2735
Fig 4. (A) A linear representation of the mature lngA protein indicating the ‘hot-spots’ of amino acid
sequence variation which are clustered within the αβ-loop and D-region (β3&4) of the molecule. (B)
A lngA homology model showing the regions of clustered variation adapted from Kolappan et al3.
D-region
αβ-loop
B
1 54 105 132 195 206
α-helical stalk αβ-loop β-strands 1 & 2 D-region (β-strands 3 & 4)
β5
N C
S S
V/K
(56)
D/S
(62)
N/S
(65) S/A
(68)
D/N
(69)
G/S/N
(76)
G/K
(77)
T/Q
(115)
T/Q
(159)
T/A
(161)
N/D
(165
)
V/I
(148)
T/N
(150)
T/A/S
(170)
T/G
(171) T/N/D
(172)
V/I
(173)
S/T
(176)
T/N
(179)
N/D/G
(180)
T/K
(183)
E/D
(188) K/R
(189)
G/S
(192)
T/Q
(197)
Variation ‘hot-spot’
β-strands
α-helical stalk
A
CS1CS3
67%
CS2CS3
20%
CS2CS3
39%
Disclaimer:
The views expressed in this poster are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. This work was supported by the U. S. Army Medical Research and Materiel Command Award 6000 RAD1 DA2 A0307 (to S. J. S.) and the USUHS/Henry M. Jackson Foundation for the Advancement of Military Medicine
Grant G186KI (to S. J. S.). S.J.S. is a military service member. This work was prepared as part of his official duties. Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.